JP4858974B2 - Method to improve plant productivity by chloroplast engineering - Google Patents

Description

  The present invention relates to a transformed plant having high photosynthetic activity and particularly excellent fixation of carbon dioxide.

  Plants carry out photosynthesis, fix carbon dioxide in the atmosphere, and synthesize sugar and organic matter that are the energy source of living organisms. In plants, the process of fixing atmospheric carbon dioxide and synthesizing sugar from carbon dioxide is called the Calvin cycle. The Calvin cycle does not require light energy and is divided into the following two stages. In the first stage, 3-phosphoglycerate (PGA) is synthesized from ribulose-1,5-bisphosphate (RuBP) and carbon dioxide, and this is further reduced to synthesize glyceraldehyde-3-phosphate (GAP). Is a process. In the second stage, a part of the synthesized GAP is used for the synthesis of sugar (photosynthesis product), and the remaining GAP is changed from fructose 1,6-bisphosphate (FBP) to fructose 6-phosphate (F6P), cedoheptulose. This process is regenerated to RuBP via 1,7-bisphosphate (SBP), sedheptulose 7-phosphate (S7P), ribose 5-phosphate, and the like. At this time, synthesis of PGA from RuBP, that is, incorporation of carbon dioxide into the Calvin cycle is catalyzed by ribulose-1,5-bisphosphate carboxylase / oxygenase (hereinafter abbreviated as Rubisco). In the second stage, aldolase [an enzyme that reversibly catalyzes the reaction from GAP and dihydroxyacetone phosphate (DHAP) to FBP and the reaction from DHAP and erythrose 4-phosphate (E4P) to SBP, respectively), fructose 1,6-bisphosphatase (FBPase; an enzyme that catalyzes the reaction from FBP to F6P), sedheptulose 1,7-bisphosphatase (SBPase; an enzyme that catalyzes the reaction from SBP to S7P), transketolase as a rate-limiting enzyme It is responsible for metabolic reactions.

  Many enzymes that act in the Calvin cycle are present above the levels required to maintain a continuous reaction of carbon dioxide fixation. However, while FBPase and SBPase are important rate-limiting enzymes in the Calvin cycle, their levels are known to be extremely low compared to other enzymes in the Calvin cycle (see: Miyagawa et al., Nature. Biotechnology (Nature Biotechnology 2001, 19th volume, p. 965-969).

  For this reason, a cytosolic fructose-1,6-bisphosphatase gene (cy-FBPase gene) obtained from mesophyll cells peculiar to plant leaves is permanently and specifically expressed as a transgenic plant that enhances photosynthetic ability. A vector having a promoter and a transgenic plant transformed with the vector have been reported (WO 98/18940).

  A method for expressing FBP / SBPase derived from the cyanobacterium Synechococcus PCC7942 gene has also been reported. According to this method, it is known that transformed plants have a higher photosynthetic activity than wild-type strains and promote growth (see: JP 2000-253768; Miyagawa et al., Nature -Biotechnology (Nature Biotechnology 2001, 19th volume, p.965-969).

  However, in any of the above transformants, each gene is introduced into the leaf nucleus genome by introducing a plasmid constructed using the gene into Agrobacterium tumefaciens and infecting the leaf disk. For this reason, the expression protein of the gene introduced into the plant had a low probability of transferring to the chloroplast.

  In addition, introduction of a heterologous gene into the nuclear genome may cause the introduced artificially modified gene to spread to the environment by crossing or mating. Furthermore, the expression of the gene thus introduced is unstable, and the expression level and thus the effect varies greatly from plant to plant.

  There are about 100 chloroplasts per adult cell, and there are 100 copies of chloroplast genome genes per chloroplast (Archives of Biotechnology and Biophysics (Archives). of Biotechnology and Biophysics), 1996, 334, p. 27-36; Bendich, AJ, BioEssays, 1987, 6, 279. -282).

  This means that if one copy of a foreign gene is inserted into the chloroplast genome, there will be 10,000 copies per cell in the transformant, and high expression of the transgene can be expected due to the large number of copies. (See Mariga, P., Trends in biotechnology, 1993, 11, p. 101-107).

  Furthermore, since gene introduction into chloroplasts utilizes homologous recombination, the position effect seen at the time of insertion into the nucleus does not occur, and stable gene expression is performed. In addition, since chloroplasts are maternally inherited, gene transfer into chloroplasts is considered to have many advantages, such as prevention of scattering of the introduced gene into the environment through pollen.

  An expression vector capable of highly expressing a target protein in a chloroplast, a transformed chloroplast transformed with the expression vector, and a plant having the transformed chloroplast are known. This expression vector is characterized by having a psbA promoter and a ribosome binding site upstream of the translation start point of the gene encoding the protein. The purpose of this method is to produce a protein having pharmacological activity, a protein useful as a material for the pharmaceutical industry, etc. using a plant instead of production by a microorganism. In this example, expression of the protein has been confirmed in plants transformed with the gene for green fluorescent protein (Maliga, P., Trends in biotechnology). 1993, Vol. 11, p. 101-107).

  However, in this document, there is no description about improving photosynthetic activity or carbon dioxide fixation in plants, nor is there any description about FBPase or SBPase, which is the rate-limiting enzyme of the Calvin cycle, or their genes. .

  An object of the present invention is to produce a transgenic plant that has higher photosynthetic activity and promotes growth than a wild strain by expressing a gene of an enzyme involved in photosynthesis, particularly in the Calvin cycle, in higher plants. It is. More specifically, the gene of an enzyme that controls the Calvin cycle reaction is introduced into chloroplast DNA to produce a plant having a transformed chloroplast with enhanced photosynthetic ability.

  The present inventors have found a transformation technique capable of reliably expressing a protein having FBPase and / or SBPase in the chloroplast of a higher plant. It was also found that the transformed plant not only has high photosynthetic activity, but also grows into a plant having a larger plant body. The present invention has been completed by repeating various studies based on these findings.

That is, the present invention
(1) A gene recombination vector having an expression cassette that enhances photosynthetic activity, comprising a DNA fragment containing a gene encoding a protein having FBPase activity and / or SBPase activity between a rubisco large subunit and an acetyl CoA carboxylase subunit gene ,
(2) The vector according to (1) above, wherein the protein having FBPase activity is any of the following:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing;
(B) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in SEQ ID NO: 1 in the sequence listing and having FBPase activity; or (c) a sequence number in the sequence listing A protein having at least 60% homology with the amino acid sequence according to 1 and having FBPase activity;
(3) The vector according to (1) above, wherein the gene encoding a protein having FBPase activity is a gene consisting of any of the following DNAs:
(A) DNA comprising the base sequence set forth in SEQ ID NO: 2 in the sequence listing;
(B) DNA encoding a protein having a base sequence in which one or several bases are deleted, substituted, added or inserted and having FBPase activity in SEQ ID NO: 2 in the sequence listing; or (c) DNA consisting of a base sequence encoding a protein that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence set forth in SEQ ID NO: 2 in the sequence table; or ( d) DNA consisting of a base sequence encoding a protein having at least 60% homology with the DNA consisting of the base sequence set forth in SEQ ID NO: 2 in the sequence listing and having FBPase activity;
(4) The vector according to (1) above, wherein the protein having SBPase activity is any of the following:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing;
(B) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted and having SBPase activity in SEQ ID NO: 3 in the sequence listing; or (c) SEQ ID NO: in the sequence listing A protein having at least 60% homology with the amino acid sequence according to 3, and having SBPase activity;
(5) The vector according to (1) above, wherein the gene encoding a protein having SBPase activity is a gene consisting of any of the following DNAs;
(A) DNA comprising the base sequence set forth in SEQ ID NO: 4 in the sequence listing;
(B) DNA encoding a protein having a base sequence in which one or several bases are deleted, substituted, added or inserted in SEQ ID NO: 4 in the sequence listing and having SBPase activity;
(C) consisting of a base sequence encoding a protein that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 4 in the sequence listing and has SBPase activity DNA; or (d) DNA consisting of a base sequence encoding a protein having at least 60% homology with the DNA consisting of the base sequence set forth in SEQ ID NO: 4 of the Sequence Listing and having SBPase activity,
(6) The vector according to (1) above, wherein the protein having FBPase activity and SBPase activity is any of the following:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 5 in the Sequence Listing;
(B) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in SEQ ID NO: 5 in the sequence listing, and having FBPase activity and SBPase activity; or (c) sequence listing A protein having at least 60% homology with the amino acid sequence of SEQ ID NO: 5 and having FBPase activity and SBPase activity,
(7) The vector according to (1) above, wherein the gene encoding a protein having FBPase activity and SBPase activity is a gene comprising any of the following DNAs:
(A) DNA comprising the base sequence set forth in SEQ ID NO: 6 in the sequence listing;
(B) a DNA encoding a protein having a base sequence in which one or several bases are deleted, substituted, added or inserted in SEQ ID NO: 6 in the sequence listing and having FBPase activity and SBPase activity;
(C) a base that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence set forth in SEQ ID NO: 6 in the sequence listing and encodes a protein having FBPase activity and SBPase activity DNA comprising a sequence; or (d) from a base sequence encoding a protein having at least 60% homology with a DNA comprising a base sequence set forth in SEQ ID NO: 6 in the sequence listing and having FBPase activity and SBPase activity. DNA,
(8) The above (1) to (7), wherein the expression cassette has a ribosome binding site upstream of the translation start point of a DNA fragment comprising a gene encoding a protein having FBPase activity and / or SBPase activity. A vector according to any one of
(9) In the above (8), the expression cassette has a terminator downstream of a DNA fragment containing a promoter and a gene encoding a protein having FBPase activity and / or SBPase activity upstream of the ribosome binding site. The described vector,
(10) The vector according to (9) above, wherein the promoter and terminator are a tobacco chloroplast-derived promoter and terminator,
(11) The vector according to any one of (1) to (10) above, wherein each of the rubisco large subunit gene and the subunit gene of acetyl CoA carboxylase is a tobacco-derived gene,
(12) Translation of the above DNA fragment having a DNA fragment containing a gene encoding a protein having FBPase activity and / or SBPase activity between a tobacco-derived large Rubisco subunit gene and a subunit gene of acetyl CoA carboxylase A ribosome binding site upstream of the start point; a tobacco-derived promoter between the rubisco large subunit gene and the ribosome binding site; and between the acetyl-CoA carboxylase subunit gene and the DNA fragment. A recombinant vector having an expression cassette having a terminator derived from tobacco,
(13) A transformed chloroplast in which the vector according to any one of (1) to (12) is introduced into a chloroplast,
(14) A plant having the transformed chloroplast according to (13),
(15) The FBP / SBP gene is introduced and expressed in the chloroplast genome of a higher plant using the plant according to (14) above, wherein the plant is tobacco, and (16) the chloroplast transformation technique. A plant that has increased FBPase activity more than twice the original plant,
About.

  Further, the present invention provides a plant having a transformed chloroplast by inserting a DNA fragment containing a gene encoding a protein having FBPase activity and / or SBPase activity into a non-coding region between chloroplast DNA genes. Relates to the method of production.

  The vector of the present invention can reliably introduce a protein having FBPase activity and / or SBPase activity into chloroplasts of higher plants. A plant transformed with the vector of the present invention has enhanced expression of a protein having FBPase activity and / or SBPase activity, which are rate-limiting enzymes of the calbin cycle, and thus has a higher photosynthetic ability than a wild strain. As a result, the transformed plant of the present invention can increase the ability to synthesize sugar and starch as compared to the wild type. Moreover, the transformed plant of the present invention has a large height, a large leaf area, a thick stem, and can grow quickly. Therefore, cultivation of a transformed plant using the vector of the present invention can be a very effective means for producing a fast-growing, high-yield plant.

  In the transformed plant of the present invention, since a gene encoding a protein having FBPase activity and / or SBPase activity is directly introduced into the chloroplast genome instead of the nuclear genome, there is a risk of spreading by the pollen of the introduced gene. Absent. That is, there is no concern about environmental pollution such as pollen being scattered over a wide area by wind or insects, such as a plant having a gene introduced into the nucleus. Moreover, expression is stable among the transformants. In addition, the transformed plant of the present invention directly introduced into the chloroplast genome has an increased ability to synthesize sugar and starch, and the height and leaves become larger compared to the transformed plant into which the gene has been introduced into the nucleus. Can grow into early, high yielding plants.

  Recombinant DNA technology can be used to improve the function of photosynthesis, which is the primary metabolism of higher plants, and to increase early harvest and yield, so it can be an extremely important technology in responding to the future food crisis.

  In addition, the transgenic plant of the present invention has an enhanced rate-limiting enzyme in the Calvin cycle that plays an important role in carbon dioxide fixation in photosynthesis. For this reason, since the transformed plant of this invention can raise the uptake | capture rate of the carbon dioxide in air | atmosphere, and can reduce the carbon dioxide density | concentration in air | atmosphere, cultivation of this plant can also contribute to suppression of global warming.

  The protein having FBPase activity and / or SBPase activity used in the present invention is a protein that can be a rate-limiting enzyme of the Calvin cycle. The protein may have any enzyme activity of FBPase and / or SBPase, or may have both enzyme activities. In particular, in higher plants, in a series of reactions in the Calvin cycle, a protein having an enzymatic activity of SBPase that can be a pacemaker enzyme that governs the overall rate of the reaction, and a protein having both FBPase and SBPase activities (hereinafter referred to as FBP). / Abbreviated as SBPase).

  Examples of the protein having FBPase activity include the amino acid sequence represented by SEQ ID NO: 1. Moreover, as a protein which has SBPase activity, the amino acid sequence shown by sequence number 3 can be mentioned, for example. Examples of the protein exhibiting FBP / SBPase activity include the amino acid sequence of cyanobacterial FBP / SBPase represented by SEQ ID NO: 5. The protein having FBPase activity and / or SBPase activity used in the present invention has an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted from the above amino acid sequences, and Also included are proteins each having FBPase activity, SBPase activity, or FBP / SBPase activity. Furthermore, the protein having FBPase activity and / or SBPase activity used in the present invention is a protein having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 1, 3 or 5, preferably 80 % Or more homologous protein, more preferably 90% or more homologous protein, more preferably 95% or more homologous protein, and FBPase activity, SBPase activity, or FBP / SBPase, respectively. Proteins having activity are also included.

  In the present specification, when “amino acid sequence” is referred to as “homologous”, the primary structures of proteins are compared and used to mean the degree of coincidence of amino acid residues constituting each sequence between sequences.

  In addition, regarding the amino acid sequence, when “one or several (about 2 to 6) amino acids are deleted, substituted, added or inserted”, by a well-known technical method such as site-directed mutagenesis, It means that a naturally occurring number has been deleted, substituted, added or inserted.

  As a DNA fragment containing a base sequence encoding a protein having FBPase activity and / or SBPase activity used in the present invention, DNA encoding each enzyme of FBPase, SBPase, FBP / SBPase and the active site of each enzyme The DNA which codes the protein which has. Examples of the base sequence encoding a protein having FBPase activity include the DNA sequence shown in SEQ ID NO: 2. Examples of the base sequence encoding a protein having SBPase activity include the DNA sequence shown in SEQ ID NO: 4. Examples of the base sequence encoding a protein having FBP / SBPase activity include the DNA sequence shown in SEQ ID NO: 6. In the DNA fragment encoding the protein having FBPase activity and / or SBPase activity used in the present invention, one or several bases are deleted from the DNA sequence shown in SEQ ID NO: 2, 4 or 6 above. DNA encoding a protein having a substituted, added or inserted base sequence and having FBPase activity, SBPase activity, or FBP / SBPase activity is included. With respect to the base sequence, when “one or several bases are deleted, substituted, added or inserted”, it is a number that can be naturally generated by a well-known technical method such as site-directed mutagenesis (1 It means that (~ several) bases have been deleted, substituted, added or inserted.

  In addition, the DNA fragment encoding the protein having FBPase activity and / or SBPase activity used in the present invention is a DNA comprising each DNA sequence shown in SEQ ID NO: 2, 4 or 6 and a complementary base sequence. And DNA comprising a base sequence encoding a protein that hybridizes with each other under stringent conditions and has FBPase activity, SBPase activity, or FBP / SBPase activity, respectively. DNA that can hybridize under stringent conditions means DNA obtained by using the above-mentioned DNA as a probe and using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. The stringent conditions are an SSC solution having a salt concentration of about 0.1 to 2 times (the composition of the SSC solution having a concentration of 1 consists of 150 mM sodium chloride and 15 mM sodium citrate), and a temperature of about 65 ° C. Hybridization conditions in terms of degree.

  Furthermore, the DNA fragment encoding the protein having FBPase activity and / or SBPase activity used in the present invention has at least 60% homology with each DNA sequence shown in SEQ ID NO: 2, 4 or 6. And DNA comprising a base sequence encoding a protein each having FBPase activity, SBPase activity, or FBP / SBPase activity. The DNA having homology is a DNA having at least about 60% homology, preferably about 80% or more homology, more preferably about 90% or more homology under highly stringent conditions. DNA having sex, more preferably DNA having homology of about 95% or more. The high stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C. Say. In particular, the most preferable conditions are when the sodium concentration is about 19 mM and the temperature is about 65 ° C.

  Hereinafter, a DNA fragment encoding a protein having FBPase activity and / or SBPase activity, and the above hybridizing DNA and homologous DNA are also referred to as a transgene.

  The expression cassette of the present invention has a gene of chloroplast DNA [for example, on the 5′- and 3′-side of the transgene to be surely introduced by homologous recombination into chloroplast DNA. trnG (tRNA-Gly (GCC)), trnV (tRNA-Val (GAC)), trnfM (tRNA-fMet (CAU)), rbcL gene, accD gene, trnI (tRNA-Ile (GAU)) and trnA (tRNA- Ala (UGU)), 3′rps12 (ribosomal protein S12 exon-3) gene, trnV (tRNA-Val (GAC)), etc.]] and a base sequence that forms a complementary base pair is added. The base sequence that forms a complementary base pair can be preferably used as long as it is a sequence consisting of about 500 to 1500 base sequences having a homologous part that forms a complementary base pair with a chloroplast DNA gene. Such a base sequence may be substantially the same sequence as the chloroplast DNA gene, substantially the same sequence as the partial sequence of the chloroplast DNA gene, or substantially the same as the chloroplast DNA gene. Examples include a complementary base sequence to a sequence containing the same sequence.

  Further, it has a base sequence of about 1000 to 1500 from the introduction site of the introduced gene, and is a chloroplast DNA gene (for example, trnG, trnfM, rbcL gene, accD gene, trnI, trnA, 3'rps12 gene, trnV Etc.), the gene sequence is not limited to the gene sequence of the chloroplast DNA.

  At this time, it is important that the base sequence of the chloroplast DNA does not change except that a foreign gene is introduced. The base sequence of chloroplast DNA into which a foreign gene is introduced has already been registered and disclosed in the NCBI database (registration number: NC 001879). The position where the introduced gene is introduced into the chloroplast DNA is preferably between the trnG and trnfM genes of the chloroplast DNA, between the rbcL gene and the accD gene, between trnI and trnA, and the 3'rps12 gene. Non-coding regions between trnV and a sufficient distance from each gene are desirable. The sufficient distance is at least 50 bases from the gene, preferably about 100 to 1000 bases, more preferably about 200 to 500 bases. The non-coding region may be any non-coding region on chloroplast DNA.

  Hereinafter, an expression cassette using the rbcL gene and the accD gene will be described in more detail.

The rbcL gene constituting an expression cassette that enhances photosynthetic activity is a Rubisco gene encoded in the chloroplast genome. Rubisco is a key enzyme that catalyzes the initial CO ₂ fixation reaction (carboxylase reaction) in the photosynthetic CO ₂ fixation reaction circuit (calvin cycle), and becomes the rate-limiting of metabolism in the circuit. The enzyme also catalyzes a reaction for fixing oxygen (O ₂ ) (oxygenase reaction). As the rbcL gene derived from chloroplasts in the present invention, the rbcL gene derived from tobacco chloroplasts can be preferably used.

  The accD gene constituting an expression cassette that enhances photosynthetic activity is an acetyl CoA carboxylase gene encoded by the chloroplast genome. Acetyl CoA carboxylase is an enzyme involved in fatty acid synthesis in plants. As the accD gene derived from chloroplasts in the present invention, an accD gene derived from tobacco chloroplasts can be preferably used.

  By using an expression cassette having a chloroplast-derived rbcL gene and a chloroplast-derived accD gene, a gene encoding a protein having FBPase activity and / or SBPase activity is incorporated into chloroplasts by homologous recombination. There is an advantage that the expression level of the protein having FBPase activity and / or SBPase activity in the chloroplast is increased.

  The rbcL gene and the accD gene do not need to use the full length thereof. For example, the non-coding region between the rbcL gene and the accD gene has a length of about 1000 to 1500 base pairs from the introduction position of the introduced gene to the rbcL gene side or the accD gene side, respectively. Any sequence that can homologously recombine with a gene may be used.

  Moreover, it is preferable that the expression cassette which raises photosynthesis activity has a ribosome binding site upstream of the translation start point of the DNA fragment containing the gene which codes the protein which has FBPase activity and / or SBPase activity. By placing a ribosome binding site upstream of the DNA fragment, the protein can be highly expressed. The ribosome binding site may be adjacent to or upstream of the translation start point of the gene encoding the protein, but is preferably about 7 to 11 bases upstream of the translation start point, and about 9 bases upstream. More preferably. Such a ribosome binding site may have a base sequence known per se that is known to be capable of binding to a ribosome, but an SD sequence is preferred. The SD sequence is an abbreviation for Shine-Dalgarno sequence and is a segment consisting of 4 to 7 nucleotides, and its base sequence is a part or all of 5′-AGGAGGU-3 ′ (SEQ ID NO: 18).

  The expression cassette that enhances photosynthesis activity preferably further has a plant cell-derived promoter upstream of the ribosome binding site. As long as the promoter is upstream of the ribosome binding site, it may be adjacent to the ribosome binding site or may be about 1 to 30 bases upstream. Examples of the promoter include an elongation factor 1α gene promoter (EF1α promoter), 35S promoter, psbA promoter, PPDK promoter, PsPAL1 promoter, PAL promoter, UBIZM1 ubiquitin promoter, rrn promoter and the like. Among them, a chloroplast-derived promoter is preferable, and a tobacco chloroplast-derived promoter is more preferable. For example, a tobacco chloroplast-derived psbA promoter described in SEQ ID NO: 7 in the Sequence Listing can be particularly preferably used.

  It is preferable that the expression cassette for enhancing the photosynthetic activity has a plant-derived terminator between a DNA fragment containing a gene encoding a protein having FBPase activity and / or SBPase activity and the accD gene. As long as the terminator is downstream of the DNA fragment, the terminator may be adjacent to the DNA fragment or may be about 1 to 30 bases downstream. Examples of the terminator include 35S terminator, rps16 terminator, CaMV35S terminator, ORF25polyA transcription terminator, PsbA terminator and the like. Among them, a terminator derived from chloroplast is preferable, a terminator derived from tobacco chloroplast is more preferable, and an rps16 terminator derived from tobacco chloroplast is most preferable, for example, derived from tobacco chloroplast described in SEQ ID NO: 8 in the Sequence Listing. Rps16 terminator can be preferably used.

  The expression cassette preferably has a gene for identifying a gene recombinant. The gene for identifying the gene recombinant is not particularly limited, and a gene known per se may be used. Examples of such genes include various drug resistance genes (aadA) and genes that complement the auxotrophy of the host. More specifically, for example, ampicillin resistance gene, neomycin resistance gene (G418 resistance), chloramphenicol resistance gene, kanamycin resistance gene, spectinomycin resistance gene, URA3 gene and the like can be mentioned. More specifically, for example, the spectinomycin resistance gene described in SEQ ID NO: 9 in the sequence listing can be preferably used. Further, a promoter for recognizing the gene (hereinafter abbreviated as aadA promoter) and a terminator for the gene (hereinafter abbreviated as aadA terminator) are arranged upstream and downstream of the gene, respectively. preferable. As the aadA promoter and aadA terminator, the above-described plant-derived promoter and terminator can be preferably used, but the rrn promoter and psbA terminator are particularly suitable. The aadA promoter / aadA / aadA terminator is sometimes referred to as an aadA cassette.

  The aadA cassette for identifying a gene recombinant is preferably arranged between the rbcL gene and a promoter upstream of the ribosome binding site.

  The expression cassette used in the vector of the present invention comprises, from the 5 ′ side, a DNA fragment, a terminator, and an accD containing a gene encoding a protein having rbcL gene, aadA cassette, promoter, ribosome binding site, FBPase activity and / or SBPase activity. It is preferably constructed in gene order. Each DNA may be continuous, or for example, an intron sequence may be inserted between each DNA.

  The gene recombination vector in the present invention can be prepared, for example, by the following steps.

First, a process for producing a pLD6 vector. Such a vector can be easily prepared by the method described in [Step 1] of the Examples. The entire base sequence of pLD6 is shown in SEQ ID NO: 10. A constructed gene group is inserted into the NotI and SalI cleavage sites of pLD6. The constructed gene group consists of (a) a multi-cloning region consisting of a base sequence represented by SEQ ID NO: 11 (located at 3698-3748 in SEQ ID NO: 10), and a tobacco leaf green represented by SEQ ID NO: 7 upstream thereof. A psbA promoter derived from the body (located at 3569-3701 in SEQ ID NO: 10) and an rps16 terminator derived from the tobacco chloroplast represented by SEQ ID NO: 8 downstream of the multicloning region (3755-3913 in SEQ ID NO: 10). AbA gene (SEQ ID NO: 10) which is a spectinomycin resistance gene represented by SEQ ID NO: 9 as a gene for identifying a gene recombinant (b) upstream of the group In the middle of 2369-3173), and the rrn pro from tobacco chloroplast represented by SEQ ID NO: 12 upstream of the aadA gene. And a psbA terminator derived from tobacco chloroplast represented by SEQ ID NO: 13 downstream of the aadA gene (located at 3175-3564 in SEQ ID NO: 10) have. A gene encoding a protein having FBPase activity and / or SBPase activity is inserted into the restriction enzyme recognition site (BglII, SphI, ClaI and EcoRI) of the multicloning region. More specifically, the SphI and EcoRI cleavage sites of the multicloning region of the pLD6 vector, for example, encode a spinach-derived FBPase shown in SEQ ID NO: 2 or a spinach-derived SBPase shown in SEQ ID NO: 4. A gene or a gene encoding FBP / SBPase represented by SEQ ID NO: 6 derived from cyanobacteria is inserted. In this case, the nucleotide sequence (5′-aggag-3 ′) at positions 13 to 17 of SEQ ID NO corresponds to the SD sequence and serves as a ribosome binding site. Hereinafter, a constructed gene group into which a gene encoding a protein having FBPase activity and / or SBPase activity is inserted is referred to as an FBP / SBP gene group, and a pLD6 vector into which the gene group is inserted is referred to as pLD6-FBP / SBP.

  Next, pLD6-FBP / SBP is introduced into an appropriate host cell, the host cell is cultured, and the FBP / SBP gene group is cloned.

  Host cells can be appropriately selected from host cells known per se. Specific examples include prokaryotes such as Escherichia and Bacillus, eukaryotes such as yeast and filamentous fungi, plant cells, and animal cells. It is done. Moreover, the culture conditions for the host cells may be in accordance with the conditions commonly used in the art depending on the type of host cell. Further, whether or not a gene encoding a protein having FBPase activity and / or SBPase activity has been successfully introduced into the cloned gene can be easily determined based on a selection marker possessed by pLD6-FBP / SBP.

  Next, it is a step of producing a pLD200 vector. Such a vector can be easily prepared by the method described in [Step 2] of the Examples. From the recombinant gene cloned using pLD6-FBP / SBP of the previous step, the FBP / SBP gene group was excised using NotI and SalI, and the excised gene group was extracted from the polylinker NotI and SalI of pLD200. Insert at the cutting site. The entire base sequence of pLD200 is set forth in SEQ ID NO: 14. The polylinker has a base sequence represented by SEQ ID NO: 17 (located at 2125-2145 of SEQ ID NO: 14) and has a plurality of restriction enzyme sites (NotI, NheI and SalI). The pLD200 vector is a tobacco chloroplast-derived rbcL gene (located at 423-1856 of SEQ ID NO: 14) consisting of a polylinker and a base sequence represented by SEQ ID NO: 15 upstream thereof, and SEQ ID NO: 16 downstream thereof. A vector characterized by having an expression cassette consisting of an accD gene derived from tobacco chloroplasts (located at 2624-3328 of SEQ ID NO: 14) having the nucleotide sequence represented by The pLD200 vector in which the FBP / SBP gene group is thus inserted is referred to as pLD200-FBP / SBP.

  The vector is a cloning vector known per se, a polylinker having a plurality of restriction enzyme sites (preferably a gene having a nucleotide sequence represented by SEQ ID NO: 17), and an rbcL gene derived from tobacco chloroplasts upstream of the polylinker. And by inserting an accD gene derived from tobacco chloroplast downstream of the polylinker.

  The pLD200-FBP / SBP prepared as described above is introduced into a host cell to prepare a transformant. At this time, the host cell is preferably a plant cell, more preferably a chloroplast, and even more preferably a tobacco chloroplast. In this way, by using plant cells, especially chloroplasts as host cells, the protein encoded by the introduced gene can be highly expressed, and further, prevention of scattering to the environment via pollen of the introduced gene is prevented. There are advantages such as being able to.

  As a method for transforming by introducing pLD200-FBP / SBPase into a host cell, particularly a chloroplast, a known method may be used. As such a method, for example, a particle gun in which the expression vector is applied to very fine particles of gold or tungsten, the particles to which the expression vector is attached is injected into a host cell with an explosive or high-pressure gas, and the expression vector is introduced. Law. Among them, a system for gene introduction into chloroplasts of higher plants is a method by particle gun (Svab, Z., Hajdukiwicz, P., and Mariga, P., Proc. Natl. Acad. Sci. USA, 1990, No. 1). 87, p. 8526-8530) or PEG-based techniques (Golds, T., Maliga, P., and Koop, H.-U., Bio / Technol., 1993, 11, p. 95-). 97) is preferably used.

  The plant having the above-mentioned transformed chloroplast according to the present invention can be obtained by a method known per se. Here, the plant is not particularly limited, but higher plants are preferred, plants with established chloroplast transformation systems are more preferred, and examples include tobacco, rice, potato, rapeseed, and lettuce, and tobacco is particularly preferred. preferable. As tobacco, Nicotiana acminate, Nicotiana alata, Nicotiana attenua, Nicotiana clevelandi, Nicotiana clevelandi, Nicotiana clevelandi (Nicotiana forgettiana), Nicotiana gossei, Nicotiana glauca, Nicotiana glutinosa, Nicotiana langusdolf Dorfii), Nicotiana longiflora, Nicotiana obtusifolia, Nicotiana paniculata, Nicotiana paniculata, Nicotiana pulabagiholia Nicotiana repanda, Nicotiana rustica, Nicotiana sanderae, Nicotiana suaveolens, Nicotiana silves Examples include Nicotiana sylvestris, Nicotiana tabacum, Nicotiana tomentosa, Nicotiana tomentosiformis, and the like. Of these, Nicotiana Rustica and Nicotiana Tabacam are preferable. In particular, Nicotiana tabacum is preferable, and “Burley species”, “Yellow species (Virginia species)”, “Native species”, and “Orient species” among Nicotiana tabacam are particularly preferable.

  The said plant can be grown on the well-known conditions according to the plant.

  The above-described genetic engineering or biotechnological operations are described in commercial experiments, for example, Molecular Cloning, Cold Spring Harbor Laboratory, published in 1982, published in 1989. It can be easily performed according to the method described in Molecular Cloning, 2nd edition (Molecular Cloning, 2nd ed.) Cold Spring Harbor Laboratory, etc.

  The vectors pLD6 and pLD200 used in the process of constructing the vector for gene introduction into the tobacco chloroplast genome according to the present invention are disclosed in Japanese Patent Application No. 2001-083569.

  Specific examples will be described below in more detail, but the present invention is not particularly limited thereto.

In addition, the meaning of each abbreviation used in an Example is as follows.
S. 7942: Synechococcus PCC 7942
LB medium: Luria-Bertani medium NaCl: sodium chloride

Preparation of gene recombinant [Step 1] pLD6-S. Preparation of 7942FBP / SBPase S. cerevisiae represented by SEQ ID NO: 2 in the sequence listing. 7942FBP / SBPase gene (fbp / sbp) was inserted between the restriction enzyme SphI and EcoRI sites of the vector pLD6 having the psbA promoter (PpsbA) that can be expected to be highly expressed in tobacco chloroplasts. 7942FBP / SBPase was prepared. This pLD6-S. 7942FBP / SBPase was introduced into E. coli according to a conventional method. This Escherichia coli was cultured in LB medium supplemented with spectinomycin at 37 ° C. for 16 hours, and Escherichia coli into which such a gene was introduced was selected. The selected Escherichia coli is cultured under the same conditions, then collected by centrifugation, and pLD6-S. 7942FBP / SBPase (plasmid DNA) was purified. The composition in 1 L of LB medium is 10 g tryptone, 5 g yeast extract, and 5 g NaCl.

[Step 2] pLD200-S. Preparation of 7942 FBP / SBPase]
The pLD6-S. After treating 7942FBP / SBPase with restriction enzymes NotI and SalI, S. A fragment containing 7942FBP / SBPase was inserted between NotI and SalI sites of the chloroplast transformation vector pLD200 containing part of the rbcL gene and part of the accD gene upstream of NotI and downstream of SalI. PLD200-S. 7942FBP / SBPase was prepared. This pLD200-S. 7942FBP / SBPase was introduced into E. coli according to a conventional method. This Escherichia coli was cultured in LB medium supplemented with spectinomycin at 37 ° C. for 16 hours, and Escherichia coli into which such a gene was introduced was selected. The selected Escherichia coli is cultured under the same conditions, then collected by centrifugation, and pLD200-S. 7942FBP / SBPase (plasmid DNA) was purified (FIG. 1).

[Step 3] Preparation of chloroplast transformant Purified pLD200-S. 7942FBP / SBPase was introduced into tobacco chloroplasts using a particle gun to prepare chloroplast transformants. Tobacco chloroplast transformation is performed according to a known method (Svab, Z., Hajdukiwicz, P. and maliga, P., Stable transformation of plastids in highger plants, Proc. Natl. Aci. (1990)).

After redifferentiation on a medium supplemented with spectinomycin, S. Six transformants (pTpsbAFS) introduced with 7942FBP / SBPase were obtained. Also in the T ₁ generation, which were generated by the self-crossed falling off of the gene did not et al observed (Fig. 2). Anti-S. As a result of Western blotting using the 7942FBP / SBPase antibody, S. cerevisiae was found only in the transformed plant (pTpsbAFS). A signal was observed at a position of about 40 kDa corresponding to the molecular mass of 7942FBP / SBPase, and it was revealed that FBP / SBPase was highly expressed (FIG. 3).

  FBPase activity was measured using plants 10 and 18 weeks after sowing. Compared to the wild strain, the transformed plant had FBPase activity that was about 10-40 times higher (FIG. 4).

With _{T 1} generation after seeding 12 weeks, it was measured photosynthetic activity by the light intensity changes in CO ₂ concentration 360ppm conditions. The result is shown in FIG. The transformants (pTpsbAFS-3 and pTpsbAFS-9) and the wild type (Wild-type) had a maximum photosynthetic rate at a light intensity of about 500 mmol / m ² / s, and thereafter maintained that rate. The photosynthetic rate of the transformant in the maximum was about twice that of the wild type.

  As a comparison, S.P. A plasmid ligated with 7942FBP / SBPase was introduced into Agrobacterium tumefaciens LBA4404 to prepare transformants (TpFS-3 and TPFS-6) infected with tobacco leaf discs. TpFS-3 and TPFS-6 had a maximum photosynthesis rate of about 1.2 to 1.3 times that of the wild type, which was much lower than that of pTpsbAFS-3 and pTpsbAFS-9. This indicates that the transformant of the present invention has significantly enhanced photosynthetic activity as compared to the wild strain and the transformed plant by the conventional method.

In addition, pTpsbAFS-3 and pTpsbAFS-9 have a light intensity of about 200 mmol / m ² / s, a maximum of the wild type, and 300 nmol / m ² / s, and the same level of photosynthesis as the maximum of TpFS-3 and TPFS-6. Showed speed. This indicates that the transformed plant of the present invention has sufficient photosynthetic activity even when the light intensity is low.

  When the growth of the transformant 18 weeks after sowing was compared with the wild strain, the growth was clearly promoted in the transformed plant compared to the wild strain, and the final growth was 1.2 to 1.3 of the wild strain. Doubled (Figures 6 and 7). In addition, the transformant had thicker stems and wild roots compared to the wild type (FIG. 8). Furthermore, the transformant after 18 weeks of growth grew to about 1.5 times the size of the wild strain.

  In this way, S.I. By introducing the 7942FBP / SBPase gene into the tobacco chloroplast genome, the photosynthetic ability of tobacco leaves could be improved. In addition, this made it possible to promote growth and increase the yield.

  For plant species other than tobacco, the plasmid pLD200-S. 7942FBP / SBP is introduced into the chloroplast by a particle gun and resistant cells are selected on a spectinomycin-supplemented medium. Plant cells expressing the 7942FBP / SBP gene can be obtained.

  For example, the plasmid is injected into a rapeseed cotyledon, a potato leaf piece, a lettuce leaf piece, a rice leaf piece or an embryonic stem cell by a particle gun, and a resistant cell is selected on a medium containing spectinomycin at an appropriate concentration. Converted plant cells can be obtained. S. By redifferentiating cells obtained by introducing and expressing the 7942FBP / SBP gene under appropriate conditions, a plant body having improved photosynthetic ability that promotes growth can be obtained. The transformation conditions for rapeseed are Transgenic Research 12 (1) p111-114 (2003), and the transformation conditions for potato are Plant Journal 19 (2) p. 209-216 (1999), the conditions for transformation of rice were described in Nature Biotechnology 17 (9) p. In 910-915 (1999), conditions for transformation on lettuce are described in Symposium of Japan Society for Plant Cell and Molecular Biology, 1 Da-10, 2004.

  Similarly, for other plant species, the above-mentioned pLD200-S. By transforming 7942FBP / SBP gene, selecting a resistant strain on a spectinomycin-added medium and redifferentiating, a transformed plant can be obtained. Selection conditions using spectinomycin can be easily determined by observing growth and redifferentiation in a medium supplemented with various concentrations of spectinomycin. Normally, it is preferable to select a concentration that is as low as possible, but at a concentration at which the wild type strain cannot grow. The regeneration condition from callus to plant can be determined using a normal method. This selection is performed using, for example, a matrix medium in which the concentration of auxin or cytokinin is changed stepwise, and the optimum conditions for redifferentiation are determined. If necessary, gibberellin or an amino acid may be added. At present, the regeneration conditions from callus to plant are determined in many plant species. By applying the chloroplast transformation techniques described above to these plants, Plants into which the 7942FBP / SBP gene has been introduced and expressed can be obtained, which has improved photosynthetic ability to promote growth. If re-differentiation becomes possible in the future for plant species for which re-differentiation conditions have not been established so far, the vector of the present invention is applied to such a plant. By introducing and expressing the 7942FBP / SBP gene, it is possible to obtain a plant whose growth is promoted by improving the photosynthetic ability.

  Plants transformed with the genetic recombinants of the present invention have high photosynthetic activity and are useful as early-growing or high-yield crops.

FIG. 1 shows the expression vector pLD200-S. It is a figure which shows 7942FBP / SBPase. FIG. 2 is a diagram showing confirmation of gene introduction by PCR. In the figure, W indicates a wild strain. FIG. 3 is a diagram showing confirmation of the transgene and the expressed protein in a plant 10 weeks after sowing. In the figure, W indicates a wild strain. FIG. 4 is a diagram showing a comparison of FBPase activity in the upper and lower leaves at 10 and 18 weeks after sowing. In the figure, the vertical axis represents FBPase activity. FIG. 5 is a graph showing photosynthetic activity at 10 weeks after sowing. FIG. 6 shows the growth rate. In the figure, the vertical axis represents the height (cm) of the plant. FIG. 7 shows a plant 18 weeks after sowing. FIG. 8 is a diagram showing stems and roots in a plant 18 weeks after sowing.

Claims (11)

Rubisco between subunit gene large subunit gene and acetyl CoA carboxylase, comprising a DNA fragment containing the gene coding psbA promoter, a protein having FBPase activity及beauty S BPase activity, and the rps16 terminator in this order,
A protein having FBPase activity and SBPase activity
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 5 in the Sequence Listing;
(B) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted and having FBPase activity and SBPase activity in SEQ ID NO: 5 in the sequence listing; or
(C) a protein having an amino acid sequence of 95% or more identical to the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing and having FBPase and SBPase activity
Or
A gene encoding a protein having FBPase activity and SBPase activity
(D) DNA consisting of the base sequence set forth in SEQ ID NO: 6 in the sequence listing;
(E) a DNA encoding a protein having a base sequence in which one or several bases are deleted, substituted, added or inserted in SEQ ID NO: 6 in the Sequence Listing and having FBPase activity and SBPase activity;
(F) a base that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 6 in the sequence listing and encodes a protein having FBPase activity and SBPase activity DNA consisting of sequences
A genetically modified vector. Expression cassette in claim 1, characterized in that it comprises a ribosome binding site downstream of the psbA promoter comprising upstream of the translation initiation point of a DNA fragment containing a gene encoding a protein having FBPase activity及beauty S BPase activity The vector described. The vector according to claim 1 or 2 , wherein the psbA promoter and the rps16 terminator are a promoter and terminator derived from tobacco chloroplasts, respectively. The vector according to claim 3, wherein the psbA promoter derived from tobacco chloroplasts consists of the base sequence of SEQ ID NO: 7. The vector according to claim 3 or 4, wherein the rps16 terminator derived from tobacco chloroplast comprises the base sequence of SEQ ID NO: 8. The vector according to any one of claims 1 to 5 , wherein the large Rubisco subunit gene and the subunit gene of acetyl CoA carboxylase are genes derived from tobacco chloroplasts . The vector according to claim 6, wherein the large Rubisco subunit gene derived from tobacco chloroplasts consists of the nucleotide sequence of SEQ ID NO: 15. The vector according to claim 6 or 7, wherein the subunit gene of acetyl-CoA carboxylase derived from tobacco chloroplast comprises the nucleotide sequence of SEQ ID NO: 16. A transformed chloroplast in which the vector according to any one of claims 1 to 8 is introduced into a chloroplast. A plant having the transformed chloroplast according to claim 9 . The plant according to claim 10 , wherein the plant is tobacco.